Al-Taweel Khaled, Dilantha Fernando W G, Brûlé-Babel Anita L
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; E-Mails:
Int J Mol Sci. 2011 Jan 18;12(1):613-26. doi: 10.3390/ijms12010613.
Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.
提取受禾谷镰刀菌Fg2感染的小麦穗的总RNA，并纯化mRNA。使用RNA转录本5'端的切换机制（SMART）技术和CDS Ill/3'引物，通过逆转录酶进行RT-PCR合成第一链cDNA。采用引物延伸聚合酶链反应构建双链cDNA，先用蛋白酶K消化，再用Sfi I消化并分级分离。收集长度超过0.5 kb的cDNA，连接到λTriplEx2载体上，随后进行λ噬菌体包装反应和文库扩增。通过常规滴度测定严格检查未扩增和扩增的cDNA文库的质量。随机挑选165个噬菌斑，使用源自载体侧翼序列的通用引物进行PCR检测。成功构建了来自受禾谷镰刀菌感染的小麦穗的高质量cDNA文库。
